Optogenetic β cell interrogation in vivo reveals a functional hierarchy directing the Ca2+ response to glucose supported by vitamin B6

Coordination of cellular activity through Ca2+ enables β cells to secrete precise quantities of insulin. To explore how the Ca2+ response is orchestrated in space and time, we implement optogenetic systems to probe the role of individual β cells in the glucose response. By targeted β cell activation/inactivation in zebrafish, we reveal a hierarchy of cells, each with a different level of influence over islet-wide Ca2+ dynamics. First-responder β cells lie at the top of the hierarchy, essential for initiating the first-phase Ca2+ response. Silencing first responders impairs the Ca2+ response to glucose. Conversely, selective activation of first responders demonstrates their increased capability to raise pan-islet Ca2+ levels compared to followers. By photolabeling and transcriptionally profiling β cells that differ in their thresholds to a glucose-stimulated Ca2+ response, we highlight vitamin B6 production as a signature pathway of first responders. We further define an evolutionarily conserved requirement for vitamin B6 in enabling the Ca2+ response to glucose in mammalian systems.

The PDF file includes: Figs. S1 to S10 Table S1 Legends for movies S1 to S23 Legends for data S1 and S2 Other Supplementary Material for this manuscript includes the following: Movies S1 to S23 Data S1 and S2

Fig. S1 .
Fig. S1.In vivo optogenetic application of NpHR3.0 decreases glucose stimulated calcium influx.(A) Images from the time-lapse recording at 6Hz of the islet before and after glucose stimulation in Tg(ins:GCaMP6s); Tg(ins:eNpHR3.0-mCherry) double-transgenic larvae.(B) Images from the islet shown in "A", during the second glucose injection and simultaneous in vivo optogenetic inhibition with a green laser (λ = 561).The optogenetic inhibition was performed by activating the laser after 7s of imaging while the glucose stimulation was delivered at 7.5s.The optogenetic inhibition was stopped after 30s of constant laser illumination.(C) Images from the time-lapse recording of the islet during the third glucose stimulation without optogenetic inhibition.(D) Traces show the normalized GCaMP6 fluorescence traces after glucose injection under normal conditions and upon light-mediated inhibition of β-cell depolarization.The red bar indicates the period of green laser exposure.(E) Quantification of the AUC reflecting 200 frames (30 s) of normalized GCaMP6 fluorescence for each condition, expressed as a fold-change with respect to the first stimulation.Each line represents an individual islet (n = 5 independent samples).1-way paired ANOVA, Tukey's correction; No inhibition (1 st injection) vs Optogenetic inhibition (2 nd injection), * p = 0.0109.Optogenetic inhibition (2 nd injection) vs No inhibition (3 rd injection), * p = 0.0267.Each data point represents an individual sample.Scale bar, 10 μm.

Fig. S2 .
Fig. S2.In vivo CheRiff temporal activation of first-responder β-cells propagates calcium signals across the islet.(A) Images from the time-lapse recording at 6Hz of an islet before, during and after the optogenetic activation with blue laser (λ = 470) of individual cells.The images belong to the experiment shown in Figure 4.The optogenetic activation was performed using a ROI-scan encompassing the area of one follower or first-responder β-cell.The numbers indicate the order in which individual cells were activated.(B) The traces show the normalized K-GECO1 fluorescence traces and the peak in calcium influx after the light-mediated activation of followers and first-responder β-cell.The blue bar indicates the period of green laser exposure.(C) Raster plots showing the normalized K-GECO1 signal for individual cells.The green arrowhead indicated the targeted cell.(D) The table shows the average time of response for individual β-cells after the glucose injection (K-GECO1 signal increase >25% above baseline, T25).The third column shows the percentage of β-cells that were co-activated by the illuminated β-cells.(E) Chart showing the proportion of β-cells grouped according to the percentage of coactivated cells (n = 39 cells from 7 independent samples).Only ~11% of β-cells can propagate the calcium across most of the cells (>75% of the cells in the plane).Scale bar, 10 μm.

Fig. S3 .
Fig. S3.Single-cell tracking in vivo shows that first-responder cells are stable for periods of 24h.(A) A cartoon showing the rationale for in vivo single-cell photoconversion and tracking.β-cellsexpress K-GECO1 and the green-to-red photoconvertible mEOS2b protein fused to histone2B (H2B).After identifying the first-responder cell, the nucleus of the cell is photoconverted from green to red by shining UV-light in a single-cell.The cell is traced for 24h.(B) Images from the time-lapse recording at 6Hz of the islet during a glucose injection at 4 dpf.The white arrowhead indicates the first-responder β-cell.(C) The traces show the cumulative normalized cytoplasmic K-GECO1 fluorescence traces and the peak in calcium influx after glucose stimulation.Glucose was injected at 5-min intervals.(D) Images from the time-lapse recording of the islet shown in "B" during the photoconversion of the first-responder cell.The photoconversion was done with a ROI-scan encompassing the area of one-cell nucleus.The white arrowhead indicates the photo-labelled β-cell.(E) The traces show the ratio of red/green fluorescence during the photo-conversion of mEOS2b for individual cells.(F) Images from the time-lapse recording of the islet during a glucose injection at 5 dpf.The white arrowhead indicates the photo-labelled cell.(G) The traces show the cumulative normalized cytoplasmic K-GECO1 fluorescence traces and the peak in calcium influx after glucose stimulation.Glucose was injected at 5-min intervals.(H) Images of the islet shown in "B-F" with numbered cells.(I) Time of response for each cell after the glucose injection at 4 and 5dpf.The photo-labelled βcell remained the first-responder cell after 24h.Scale bar = 10µm.

Fig. S4 .
Fig. S4.The speed of glucose uptake does not define the first-responder cell.(A) Cartoon representing the comparison between the calcium responses upon glucose stimulation versus the glucose uptake using the green fluorescent glucose analog 2-NBDG.(B) The fluorescence spectra of 2-NBDG and K-GECO do not overlap and can be combined in live imaging.(C) Snapshots from a time-lapse recording of the primary islet at 6Hz before and after glucose injection.The white arrow points to the β-cell that responded first to the glucose stimulus.(D) Snapshots from a time-lapse recording of the primary islet at 20Hz (50ms per frame) before and after 2-NBDG injection.The green signal corresponds to the 2-NBDG signal.(E, F) The traces show the normalized K-GECO1 and normalized 2-NBDG fluorescence traces for the calcium signal and the glucose uptake signal.FI, fluorescence intensity.(G, H) Raster plots show the normalized K-GECO1 and 2-NBDG signal for individual cells.The green arrowhead indicated the first-responder cell.(I) The numbers indicate the cells that were analyzed in "C" and "D" to quantify the individual time of response (defined as a > 25% increase in K-GECO and 2-NBDG fluorescence after injection).(J) The table shows a color key of the temporal order of response for individual cells to glucose injection (K-GECO1) and glucose uptake (2-NBDG).(K) Graph plotting each cell's temporal order of activation (K-GECO1) versus the glucose uptake (2-NBDG) from the islet shown in "I".The black line shows a linear regression and the associated R 2 value.The dotted red lines show the 95% confidence interval.Each dot represents one cell.(L)Graph showing the R 2 from 5 independent samples with an average R 2 value of 0.087 and ± SD of 0.13.(n = 5 independent samples).Data shown as mean ± SD.Scale bar = 10µm.

Fig. S5 .
Fig. S5.Characterization of pnpo + β-cells in zebrafish.(A) TSNE plot from single-cell RNA sequencing of the 2 mpf zebrafish pancreas highlighting pancreatic cell types.(B) Feature plots showing the expression of ins and pnpo in pancreatic cells.(C) Violin plot showing the lognormalized expression of ins and pnpo in β-cells.(D) Feature scatter plot highlighting the ins and pnpo expressing β-cells.The double-positive cells are marked in red.(E) Heat-map highlighting differentially expressed genes between pnpoand pnpo+ β-cells.(F) Bar plots showing the Gene Ontology (GO) and KEGG pathway analysis performed using upregulated DEGs from pnpo+ β-cells (p < 0.05)

Fig. S9 .
Fig. S9.Vitamin B6 antagonism reduces in a dose-dependent manner the first-and the second-phase responses to glucose.(A) Graphical representation of a typical calcium trace of mouse islets.T25 is the time taken from the glucose addition in the media to observing a GCAMP fluorescence increase of 25% over the baseline.The amplitude of the first-phase response is defined as the maximum value recorded during the initial increase in calcium.The peak width is defined as the time spent with a fluorescence signal greater than 25% over the baseline.During the second-phase response, the amplitudes and the peak widths are averaged.(B) The graph shows the percentage of islets that showed significant activity during the firstphase and the second-phase responses upon DMSO or 4-DP treatment.(C) T25 quantifications for controls and 4-DP treated islets.(D-E) Peak amplitude and peak width quantifications of the first-phase response of DMSO and 4-DP-treated islets.(F-G) Average peak amplitude and peak width quantifications of the second-phase response upon DMSO or 4-DP treatment.The experimental groups include 0.5 mM 4-DP, 1mM 4-DP, 2mM 4-DP, and a 1 min rinse of 2mM 4-DP (n = 31, n = 42, n = 43, n = 37, respectively from 3 mice).Data significance was analyzed by 1-way unpaired ANOVA, Tukey's correction.Panel B, **** p = 2.66E-10.Panel C, **** p = 5.06E-58.Panel D, **** p = 6.28E-77.Panel E, **** p =5.02E-67.Panel F, * p = 0.0158, ** p-value = 0.0015, *** p = 0.0001, **** p = 8.02E-53.Panel G, ** p = 0.0023, **** p = 4.21E-46.ns, not significant.Data are shown as mean ± SD.

Table S1 . General properties of commonly used optogenetic actuators
2Values obtained from reference (23) 3 Values obtained from reference(24)